Limitations of safranin ‘O’ staining in proteoglycan-depleted cartilage demonstrated with monoclonal antibodies

Summary The intensity of safranin O staining is directly proportional to the proteoglycan content in normal cartilage. Safranin O has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin O staining, in both normal and arthritic tissues. In cartilage where safranin O staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin O is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.     

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.     

Articular-cartilage proteoglycans in aging and osteoarthritis.

The composition of macroscopically normal hip articular cartilage obtained from dogs of various ages was studied. Pieces of cartilage with signs of degeneration were studied separately. In normal aging, the extraction yield of proteoglycans decreased; the keratan sulphate content of extracted proteoglycans increased and the chondroitin sulphate content decreased. The extracted proteoglycans were smaller in the older cartilage, mainly owing to a decrease in the chondroitin sulphate-rich region of the proteoglycan monomers. The hyaluronic acid-binding region and the keratan sulphaterich region were increased and the molar concentration of proteoglycan probably increase with increasing age. The degenerated cartilage had higher water content and the proteoglycans, as well as other tissue components, gave higher yields. The proteoglycan monomers from the degenerated cartilage were smaller than those from normal cartilage of the same age, and hence had a smaller chondroitin sulphate-rich region and some of the molecules also appeared to lack the hyaluronic acid-binding region. Increased proteolytic activity may be involved in the process of cartilage degeneration.     

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Monoclonal antibodies to different protein-related epitopes of human articular cartilage proteoglycans.

Monoclonal antibodies produced against chondroitinase-treated human adult cartilage proteoglycans were selected for their ability to recognize epitopes on native proteoglycans. Binding analyses revealed that four of these monoclonal antibodies (BCD-4, BCD-7, EFG-4 and KPC-190) each recognized a different epitope on the same proteoglycan molecule which represents a subpopulation of a high buoyant density (D1) fraction of human articular cartilage proteoglycans (10, 30, 50 and 60% in fetal-newborn, 1.5 years old, 15 years old and 52-56 years old cartilages, respectively). Analysis of epitope specificities revealed that BCD-7 and EFG-4 monoclonal antibodies recognized epitopes on proteoglycan monomer which are associated with the protein structure in that they are sensitive to cleavage by Pronase, papain and alkali treatment and do not include keratan sulphate, chondroitin sulphate or oligosaccharides. The BCD-4 and KPC-190 epitopes also proved to be sensitive to Pronase or papain digestion or to alkali treatment, but keratanase or endo-beta-galactosidase also reduced the immunoreactivity of these epitopes. These observations indicate that the BCD-4 and KPC-190 epitopes represent peptides substituted with keratan sulphate or keratan sulphate-like structures. The BCD-4 epitope is, however, absent from a keratan sulphate-rich fragment of human adult proteoglycan, while the other three epitopes were detected in this fragment. None of these four epitopes were detected in the link proteins of human cartilage, in the hyaluronic acid-binding region of human newborn cartilage proteoglycan, in Swarm rat chondrosarcoma proteoglycan, in chicken limb bud proteoglycan monomer and in the small dermatan sulphate-proteoglycan of bovine costal cartilage. EFG-4 and KPC-190 epitopes were not detected in human fetal cartilage proteoglycans, although fetal molecules contained trace amounts of epitopes reactive with BCD-4 and BCD-7 antibodies.     

A chondroitin sulphate proteoglycan from human cultured glial and glioma cells. Structural and functional properties.

A chondroitin sulphate proteoglycan capable of forming large aggregates with hyaluronic acid was identified in cultures of human glial and glioma cells. The glial- cell- and glioma-cell-derived products were mutually indistinguishable and had some basic properties in common with the analogous chondroitin sulphate proteoglycan of cartilage: hydrodynamic size, dependence on a minimal size of hyaluronic acid for recognition, stabilization of aggregates by link protein, and precipitability with antibodies raised against bovine cartilage chondroitin sulphate proteoglycan. However, they differed in some aspects: lower buoyant density, larger, but fewer, chondroitin sulphate side chains, presence of iduronic acid-containing repeating units, and absence (less than 1%) of keratan sulphate. Apparently the major difference between glial/glioma and cartilage chondroitin sulphate proteoglycans relates to the glycan rather than to the protein moiety of the molecule.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Proteoglycan components of the intervertebral disc and cartilage endplate: an immunolocalization study of animal and human tissues

Summary Monoclonal antibodies have been used to study the presence and distribution of various components of the proteoglycan molecule in the intervertebral disc and cartilage endplate. Link protein, hyaluronic acid binding region, keratan sulphate and chondroitin 4- and 6-sulphate have been investigated in tissues from humans and other mammals. Exposure of the carbohydrate and protein epitopes was enhanced by chondroitinase and trypsin pretreatment respectively. The degree of immunoreactivity varied with location, being greater in the nucleus pulposus than the annulus fibrosus with least reactivity in the cartilage endplate. In addition, there was increased staining in the pericellular domains, particularly in adult tissues. Areas of ectopic calcification exhibited very different immunoreactivity, depending on the type of calcium salt present. Calcium hydroxyapatite deposits showed greater staining for 8A4 (link protein), while calcium pyrophosphate deposits demonstrated greater staining for 3B3(-), 7D4(-) and 3D5 than the surrounding non-calcified matrix. Staining for chondroitin sulphate isomer epitopes 3B3(-) and 7D4(-), indicative of modified chondroitin sulphate chains, was greater in human tissues of degenerate than non-degenerate appearance. This suggests that expression of these epitopes may be an indicator of disease and subsequent reparative procedures in intervertebral disc and cartilage endplate, similar to that seen in articular cartilage degeneration.     

Studies on the in vitro incubation procedure for [35]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [³⁵S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns.The incorporation of [³⁵S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows.The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow.The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Studies on the in vitro incubation procedure for [35S]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [35S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns. The incorporation of [35S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows. The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow. The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.     

The identification and characterization of two populations of aggregating proteoglycans of high buoyant density isolated from post-natal human articular cartilages of different ages.

After chromatography on Sepharose CL-2B under associative conditions, high-buoyant-density human articular-cartilage proteoglycans were analysed biochemically and by radioimmunoassay with monoclonal antibodies to a core-protein-related epitope and to keratan sulphate. An examination of proteoglycans from individuals of different ages revealed the presence at 1 year of mainly a single polydisperse population containing chondroitin sulphate (uronic acid) and keratan sulphate. From 4 years onwards a smaller keratan sulphate-rich and chondroitin sulphate-deficient population appears in increasing amounts until 15 years. At the same time the larger population shows a progressive decrease in size from 1 year onward. By 23 years and after the proportion of keratan sulphate in the larger chondroitin sulphate-rich proteoglycan increases. Both adult proteoglycan populations are shown immunologically to aggregate with hyaluronic acid, with the smaller showing a greater degree of interaction. The larger population is richer in serine and glycine, and the smaller population contains more glutamic acid/glutamine, alanine, phenylalanine, lysine and arginine; its protein content is also higher. Whether the larger post-natal population represents a different gene product from the single polydisperse population found in the human fetus, which has a different amino acid composition, remains to be established. The smaller population, which represents approximately one-third the mass of the larger population in the adult, may represent a degradation product of the larger population, in which the hyaluronic acid-binding region and keratan sulphate-rich region are conserved.     

Separation and characterization of two populations of aggregating proteoglycans from cartilage.

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 X 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 X 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.     

The presence of a cartilage-like proteoglycan in the adult human meniscus.

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This 'cartilage-like' proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the 'cartilage-like' proteoglycan, which does not contain dermatan sulphate.     

Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo.

This study consists of (1) the extraction of proteoglycan from the human meniscus under dissociative conditions, (2) an investigation of the changes that occur in the abundance and structure of this proteoglycan with age and (3) a comparison of these findings with those for human articular-cartilage proteoglycan. Adult meniscus was found to possess proteoglycan molecules of similar size and glycosaminoglycan content to those present in cartilage, although tissue concentrations were considerably lower. In addition, age-related changes, with respect to the occurrence of keratan sulphate and the sulphation of chondroitin sulphate chains, were common to both tissues. The presence of aggregated proteoglycan was demonstrated, although specific interaction with hyaluronic acid was not conclusively shown biochemically. Differences were, however, noted in the structure of the proteoglycan between the two tissues: dermatan sulphate was found in the meniscus proteoglycan preparation and the core proteins exhibited some dissimilarities. A proteoglycan structure of this type would be compatible with its participation in meniscus elasticity, especially as the material is localized in a specific area.